OBJECTIVE: To evaluate the effects of
physical training of mother rats during pregnancy associated with a low-protein
diet offered during pregnancy and lactation on the development and growth of
the femur of their offspring.METHODS: Forty 90-day old male Wistar rats were divided into four groups:
pups of sedentary nourished mothers, pups of sedentary malnourished mothers,
pups of trained nourished mothers, and pups of trained malnourished mothers;
all groups included 10 rats. Physical training on a treadmill for 8 weeks, 5
weeks before conception and 3 weeks in the gestational period for mother rats
of pups of trained nourished mothers and pups of trained malnourished mothers.
Induction of low-protein diet to the mother rats during pregnancy and lactation
for the groups of pups of sedentary malnourished mothers and trained malnourished
mothers. After the pups were sacrificed, on the 90th day of life, we analyzed
weight, length, and femoral bone mineral content.RESULTS: Decreased body weight, femur weight, and femur length (p < 0.05)
were observed for the groups of pups of sedentary malnourished mothers and trained
malnourished mothers in comparison with to the groups of pups of sedentary nourished
mothers and trained nourished mothers, respectively. There was no difference
in bone mineral content of the femur in either of the groups.CONCLUSION: Mild of physical training on the treadmill during pregnancy
does not interfere with bone development and growth of the offspring. However,
protein malnutrition during this period and during lactation promotes permanent
damage to the bone structure of the offspring.

During pregnancy, the practice of moderate and
appropriate physical activity brings several benefits to different systems of
the maternal body.1 On the other hand, physical exercises performed
in an inadequate manner by pregnant women may impair fetal development, since
this practice may interfere with the distribution of blood flow to the placenta,
damaging the supply of oxygen and nutrients.1

It is natural for pregnant women to need a higher
carbohydrate intake, both at rest and during exercise, than non-pregnant women.2,3
After the 13th week of pregnancy, about extra 300 kcal per day are needed to
meet the metabolic needs of a pregnant woman.1,4 This extra energy
requirement is increased when the daily energy expenditure is increased due
to exercises.

On their turn, proteins are also nutrients necessary
for cell homeostasis, and when a pregnant woman has a protein deficit, known
as prenatal malnutrition, the course of the pregnancy will be affected, causing
changes in the tissues and organs of the fetus.5 Bone tissue is sensitive
to protein malnutrition due to the fact that its non-mineral bone composition
is mostly constituted by protein; therefore, it is evident that there is a relationship
between proper protein absorption and bone metabolism6,7 and that
prenatal and neonatal malnutrition will lead to disorders in bone growth.8
However, not only the period in which it occurs, but also the duration of the
nutritional deficit is an important factor to estimate the effects on skeletal
growth.9

Due to the large increase in physical activity
practice by pregnant women and high prevalence of malnutrition in developing
countries, it is important to conduct experimental studies to assess possible
consequences of both factors: effect of physical exercise and malnutrition induced
in pregnant rats on the process of bone growth of the offspring. Based on this
information, the objective of the present study was to evaluate the effects
of physical training induced in pregnant rats associated with low-protein diet
induced during pregnancy and lactation on the bone tissue growth of pups.

Methods

Experimental design

Before conception, twenty-eight adult female
albino rats (Wistar) were randomly divided into two groups: sedentary
mother (Sm) and trained mother (Tm). In order to adapt to the physical training,
the rats of Tm group performed a low intensity run (0.3 km/h-1
during 10 min) on a treadmill (motorized treadmill INSIGHT®,
1380 x 600 x 400 mm - L x W x H) for 3 consecutive days. Next, the rats accomplished
a moderate physical training program in accordance with the protocol suggested
by Fidalgo.10 The intensity used was approximately 50% of the maximum
speed reached during the performance test and 70% of VO2max (maximal
oxygen uptake). During this period, the animals in the Sm group remained in
their cages.

After 5 weeks of physical training, the rats
in both groups were mated (one male rat to three female rats). After conception
was confirmed using a vaginal smear test,11 the training protocol
was changed for the rats of the Tm group. The speed and duration of the stages
were progressively reduced, featuring a mild training, with approximately 25%
of maximum speed reached during the maximum performance test and 30% of VO2max.10

Furthermore, after pregnancy was confirmed, half
of the mother rats in each group (Sm and Tm) were offered low-protein diet (8%
casein protein) and the remaining rats received a normal protein diet (17% casein
protein) during the entire period of gestation and lactation of pups. One day
after birth, the litter was standardized in six male pups per mother.
This number seems to provide higher lactotrophic potential (Committee on Laboratory
Animal Diets).

After lactation, on the 22nd day of life of the
pups, the mothers were separated from their offspring and all animals received
the same standard diet in guinea pig cages (LABINA, Purina do Brasil). Pups
were kept in an experimental animal facility, at temperature of 23 °C ± 1, inverted
light-dark cycle of 12/12 hours and free access to water and food.

The animals' body weight was weighed daily from
birth until 90 days of life (using a scale Marte®, model S-4000,
with a sensitivity of 0.1 g). In the 90th day of life, the animals were sacrificed
by decapitation and an incision was made from the right lower abdominal region
to the knee on the same side for disarticulation and femur removal. After dissection,
femurs were fixed in buffered formalin (10 mL formalin at 37% and 27 mL of 0.1
M phosphate buffer and pH = 7.0) at a volume 50 times higher than the sample
and stored in containers. The procedures performed in the present study were
approved by the Research Ethics Committee on Animal Experiments (CEEA) of Universidade
Federal de Pernambuco (UFPE) and complied with the standards suggested by the
Brazilian Committee of Animal Experiments (COBEA).

Morphometric analysis

The weight of femurs was measured using a digital
hydrostatic balance (sensitivity for density 0.001 g; maximum capacity of 500
g, and minimum capacity of 0.02 g) (Marte®). The total bone length
was determined using a pachymeter (Western, 0.02 mm). Finally, after decalcification
carried out using nitric acid solution at 10% for 2.5 hours for the pSNm and
pTNm groups and 2 hours for the pSMm and pTMm groups, bone mineral content was
calculated using the formula suggested by Gomes et al.12

Statistical treatment

The data on body weight, femur weight, femur
length, and femur mineral content of the pSNm, pSMm, pTNm and pTMm groups of
the animals studied were statistically analyzed using the Student's t test.
Data are presented as mean ± standard deviation in a table, and p-value was
< 0.05.

Results

Weight evolution

At 21 days of life, weaning phase of the animals,
pups of the pSNm group (38.33±6.4 g) showed difference in body weight in relation
to the pSMm group (21.77±3.89 g). Likewise, pups of the pTNm group (40.5±2.88
g) showed a difference in body weight at 21 days of life compared to the pups
in the pTMm group (22.25±2.75 g). However, we did not observe any difference
in body weight between the animals of the pSNm and pTNm groups, as well as no
difference in body weight at 21 days of life between the animals of the pSMm
and pTMm groups. Therefore, only malnutrition caused changes in body weight,
since only the malnourished animals showed a difference in body weight at 21
days of life compared to the malnourished groups (Figure 1).

At 45 days of life, the body weight of the animals
in the pSNm group (174.57±8.67 g) showed a difference in body weight in relation
to the pSMm group (143±6.48 g). The same happened with the pups of the pTNm
group (173±17.34 g), which showed a difference in body weight when compared
to the pups in the pTMm group (133.6±16.74 g). On the other hand, we did not
find any difference in body weight between the animals of the pSNm and pTNm
groups, as well as no difference in body weight at 21 days of life between the
animals of the pSMm and pTMm groups. The difference in body weight persisted
in the group of malnourished animals, which occurred at 21 days of life, since
malnutrition was the only factor of change in the absolute weight of the animal
(Figure 1).

In the present study, we found that the malnutrition
induced during pregnancy and lactation of mother rats had an influence on the
body weight of pups, since the weight of the animals in the pSNm group (340.33±16.24
g), measured at 90 days of life, was higher in comparison with the values found
for the pSMm group (249.88±21.86 g). However, physical training of nourished
pregnant rats did not cause changes in the body weight of their offspring, since
there was no difference in the body weight of the pups in the pSNm and pTNm
groups (363.3±30.14 g). In both groups of pups of malnourished mothers, physical
exercise induced in the malnourished rats also did not affect the body weight
of their offspring, since the body weight of the pups in the pSMm group remained
equivalent when compared to the body weight of the offspring of the pTMm group
(305±16.80 g). Animals in the group pTNm, on their turn, had higher body weight
compared to the body weight of the offspring in the pTMm group after 90 days
(Figure 1).

Femur weight

At 90 days of life, the femur weight of the offspring
in the pSNm group (0.892±0.0661 g) proved to be greater than the femur weight
of the offspring in the pSMm group (0.81±0.058 g), which had malnutrition induced
during gestation and lactation. However, the femur weight of the animals in
the pSNm group did not show any difference in relation to the femur weight of
the animals in the pTNm group (0.9±0.0528 g), whose nourished mothers practiced
physical activity during pregnancy. The same also occurred regarding the femur
weight of the animals in the pSMm group when compared to the femur weight of
the animals in the pTMm group (0.813±0.0334 g). In the same period, the
femur weight of the pups in the pTNm group was higher than the femur weight
of the pups in the pTMm group, in which the mother rats in both groups practiced
physical activity during pregnancy (Table
1).

Femur length

At 90 days of life, the femur length, which indicates
the longitudinal growth of the bone, in the pSNm group (35.787±0.99 mm) was
higher than the length of the femur of the pups in the pSMm during gestation
and lactation (34.46±0.37 mm) The same group pSNm, on its turn, showed that
the femur length of its pups was equivalent to the femur length of the pups
in the pTNm group (35.599±0.46 mm), as well as the femur length of the offspring
in the pSMm group was also equivalent to the femur length of the offspring
in the pTMm group (34.302±0.718 mm) in the same period. The animals in group
pTNm showed higher values of femoral length when compared to the values of the
pTMm group (Table 1).

Bone mineral content

Neither the physical training during pregnancy
of mother rats nor the low-protein diet induced in mother rats during pregnancy
and lactation affected the mineral content of the femur of their pups because
there was no difference in mineral content of the femur of the pups of the pSNm
(30.386 ± 5.776 mg/cm2), pSMm (25.012 ± 6.297 mg/cm2),
pTNm (30.18 ± 6.16 mg/cm2) and pTMm groups (23.671 ± 7.775
mg/cm2) at 90 days of life (Table
1).

Discussion

The individual's growth is conditioned not only
by genetic background but also by environmental stimuli, such as, for example,
adequate supply of nutrients and practice of physical activity.1
A low-protein diet during critical periods of development severely affects all
tissues, causing harmful effects on the growth of several organs, mainly the
bone tissue in patients undergoing such aggression.13,14

In this study, protein malnutrition imposed to
the mother rats during pregnancy and lactation periods resulted in a deficit
observed until the end of the study regarding the body weight of the offspring.
Similar to these findings, Golstein & Bond15 observed that mice
that underwent protein deprivation during lactation weighed less than the control
animals, which persisted even with nutritional supplementation. This phase is
considered a period of physiological stress for the newborn, during which it
goes through several adjustments leading to a high energy expenditure.16
Thus, the infant's weight gain rate is high, and inadequate intake regarding
the quality or quantity of food may influence the reduction in body weight.17

The physical training on a treadmill carried
out by the nurturing mothers during pregnancy, on its turn, did not cause changes
in the pups' weight. Unlike our findings, a study conducted with pregnant women
who exercised during pregnancy showed that their children were born weighing
more than the children of sedentary mothers.18 Likewise, Clapp et
al.19 highlighted that adequate physical exercise throughout the
whole human pregnancy, or part of it, may contribute to the increase in fat
percentage, weight, and head circumference of children. However, the physical
training protocol used in the study by Clapp et al.19 differs from
that used in the present study: the maximum aerobic capacity corresponded from
55 to 60%, the physical training lasted for 20 min, from three to five times
a week, and the physical exercise included the use ladder or stool.

The femur weight of the offspring was assessed
at the age of 90 days of life and showed a behavior similar to that presented
by body weight, with decreased weight of the animals whose mothers were malnourished
during the critical period of development. Similarly, Boyer et al.20
demonstrated that the femur weight was also reduced according to the nutritional
deficit. Malnutrition during these important phases of development causes failure
to complete bone recovery at least until 100 days of life in rates21
and up to 6 months of life in human beings.22

Physical training during pregnancy, on its turn,
did not change the femur weight of the pups. There are no reports in the literature
demonstrating the influence of physical training during pregnancy on the bone
tissue of the offspring, making it impossible to make any comparison with the
findings of the present study.

Some studies have reported a decrease in the
femur length after induced malnutrition both in the early period (postnatal)23,24
in late (after the postnatal period). In the present study, the femur length
of the pups of malnourished mother rats was reduced at 90 days of life. The
reduction in the length of long bones, such as the femur, due to a protein deficiency
has often been attributed to a decrease in bone formation25 and a
lower rate of appositional bone growth.26 Physical training during
the gestational phase, similarly to the data of body weight and femur weight,
did not change the femur length of the pups. We expected that the stress of
training was harmful, aggravating the damage caused by the nutritional aggression
against the bone, since physical activity occurs during the formation of bone
tissue and also uses extra energy expenditure in addition to the required energy
intake during the gestational period for the development of the fetus.

In our study, we found that the time of decalcification
of malnourished femurs was reduced in 30 min with respect to the nourished femurs.
The suppression of longitudinal growth of long bones of animals caused by protein
malnutrition during lactation has been described by several studies, and most
likely occurs as a result of a quantitative decrease in bone formed than due
to a change in the mineral content.27,28 Our findings support this
hypothesis, since there was no difference in the mineral bone content among
the groups, even when being influenced by the treadmill training during pregnancy
and/or protein malnutrition of the mother during pregnancy and lactation.

The concept of "fetal programming"
suggests that the fetus can be programmed during intrauterine development for
developing diseases in adulthood, and it has been discussed as a key factor
to understand the origin of some disorders observed during adulthood.29
The basic hypothesis suggests that the fetus is programmed in the uterus to
develop many diseases in adulthood, including mainly metabolic and cardiovascular
diseases as a result of some attacks, which permanently change physiological
and metabolic processes. The specific nature of these attacks is unclear, however,
there seem be involvement of fetal growth retardation at a specific stage of
development.30

The model of experimental protein malnutrition
of our study was induced at the time of maternal depletion of reserves, which
is common during pregnancy and lactation and could have been exacerbated by
increased nutritional demand during physical training. Based on these findings,
there is stronger evidence that protein malnutrition imposed during critical
developmental periods leads to a stunted growth of the animal and that the severity
of this deficit is related to stage of life during which it was induced, which
are crucial periods for bone formation and type of impairment.23,25

Protein malnutrition during pregnancy and lactation
promotes permanent damage to the bone structure of the pups. However, the practice
of mild physical activity performed by female rats during pregnancy does not
intensify the changes in the bone structure of the malnourished offspring or
promotes changes in the bones of nourished offspring.

Acknowledgements

To Professor Carol Leandro of UFPE (Campus Vitória)
for her cooperation and assistance with the euthanasia of the rats.

This study was conducted at Universidade Federal
de Pernambuco (UFPE), Recife, PE, Brazil.
No conflicts of interest declared concerning the publication of this article.Suggested citation: Monteiro AC, Paes ST, dos Santos JA, de Lira KD,
de Moraes SR. Effects of physical exercise during pregnancy and protein malnutrition
during pregnancy and lactation on the development and growth of the offspring's
femur. J Pediatr (Rio J). 2010;86(3):233-238.